The dynamic modification of intracellular proteins by monosaccharides of O-linked N-acetylglucosamine (O- GlcNAc) plays key roles in cellular physiology and the progression of diseases which include type II diabetes, cancer, neurodegeneration, heart failure, hypertension and aging (1, 2). However, the molecular events that underlie the role of O-GlcNAc in the pathophysiology of these diseases are ill defined. In part, this is due to a lack of tools that facilitate the specific and temporal modulation of O-GlcNAc in vitro and in vivo. The goal of this study is to generate facile chemical-genetic tools that enable the spatial, temporal and dose-dependent regulation of O-GlcNAcylation in vitro and in vivo. To achieve these goals we will address the following specific aims: Specific Aim #1: Enabling tunable, reversible chemical-genetic regulation of O-GlcNAcylation. The proposed studies will tag the endogenous loci of the enzymes that add and remove O-GlcNAc with fluorescent- and affinity-tags, and introduce recombination-inducible bi-orthogonal chemical-genetic regulators. Together, these tools will enable imaging and purification of the endogenous O-GlcNAc modifying enzymes and the rapid and synthetic regulation of protein O-GlcNAcylation. Specific Aim #2: Enabling tunable tissue-specific regulation of O-GlcNAcylation in vivo. The proposed studies will generate mice in which the expression of the enzymes that add and remove O- GlcNAc can be regulated in a dose-dependent, tissue specific and temporal manner. Together, the development of the innovative tools described in this proposal will overcome current experimental limitations to O-GlcNAcylation research, facilitate studies focused on determining the biological roles of protein O-GlcNAcylation at a mechanistic level, and generate a pre-clinical murine model in which the role of O-GlcNAc in the pathophysiology of a broad range of diseases can be rapidly and specifically tested.